The invention relates to the field of biological indicators, and in particular to biological indicators for the validation of treatment processes designed to reduce the amount or activity of a contaminant in a sample. The invention further relates to methods of preparing these indicators, and to the uses thereof.
A wide variety of biological indicators are known for validating cleaning and decontamination processes. These range from relatively basic indicators, such as those that use a simple “visual score” to assess whether a process has been effective, to more sophisticated indicators that rely on thermostable kinases as reporter enzymes (WO2005/093085). These kinase-based indicators have been an important development in the biological indicator field, providing a rapid and sensitive means of process validation.
WO2005/093085 describes in detail the production and use of the kinase-based indicators referred to above. In summary, a typical indicator is prepared by adsorbing a thermostable kinase onto a solid support such as an indicator strip or dipstick. The indicator is then included with a sample (containing a contaminant) to be treated, and the indicator plus sample are subjected to a treatment process. The reduction in activity of the indicator kinase by the treatment is then correlated with the reduction in amount or activity of the contaminant. When a level of activity is determined that is known to correlate with an acceptable reduction in the contaminant, the treatment is then regarded as validated.
It has now been found that the performance of these kinase-based indicators can be significantly improved by covalently cross-linking the thermostable kinase to a biological component, wherein the biological component is a mimetic/surrogate of the contaminant. This allows the indicator to more accurately reflect the reaction of the contaminant to the treatment process, which in turn leads to improved indicator accuracy/sensitivity, and thus fewer “false” process validations.
Advantageously, the biological component may be part of a biological matrix or mixture, such as a commercially available test soil (Browne soil, Edinburgh soil etc.), blood, neurological tissue, food, culled animal material, serum, egg, mucus, or a test soil made up to meet the specific requirements of the user. In this way, the reduction in the amount/activity of the kinase is a function of the diverse properties of the matrix, which further improves the accuracy/sensitivity of the indicator.
An indicator of this type is also able to monitor the removal/inactivation of a specific component of the matrix or mixture. Advantageously, an indicator can be designed so that the thermostable kinase is linked to the most “difficult” component of the matrix to remove/inactivate (e.g., in a matrix of blood, fibrin is much more difficult to remove than haemoglobin). This provides for an extremely stringent validation of the treatment process.
The indicators described above also have the advantage of providing rapid, single step, process validations. This is in contrast to certain known validation indicators, which require multiple steps for validation and therefore require a much greater investment of time and effort. By way of example, WO00/65344, describes the use of a yeast prion as a biological indicator for a prion decontamination process. At the end of the process, the operator must, in a further step, assay the destruction of the yeast prion in order to validate the process. In contrast, the indicators described above are designed to have an indicator kinase linked directly to a biological component that mimics the relevant contaminant (e.g., prion) so that the destruction of this component is intimately linked to the loss of kinase activity. As such, these indicators are able to provide for a rapid single-step indication of process efficacy.
The invention therefore addresses the problem of providing an alternative/improved kinase-based biological indicator.
Biological Process Indicator
In a first aspect of the invention, there is provided a biological process indicator for validating a treatment process in which the amount or activity of a contaminant in a sample is reduced, wherein the indicator comprises a thermostable kinase covalently linked to a biological component, with the proviso that the biological component is not an antibody.
In one embodiment, the biological component is a mimetic or surrogate of the contaminant, and therefore reacts to the treatment process in substantially the same way as the contaminant. In another embodiment, the biological component may be the same as, but physically distinct from, the contaminant in the sample that is to be subjected to the treatment process, e.g., if the contaminant is a protein, then the biological component is also a protein; if the contaminant is a blood protein, the biological component is also blood protein; if the contaminant is a DNA molecule, then the biological component is also a DNA molecule; if the contaminant is an RNA molecule then the biological component is also an RNA molecule, etc. for each of the contaminants and biological components disclosed in this specification. In a further embodiment, the biological component may be different from the contaminant.
Examples of biological components that can be used in the indicators of the invention include proteins, nucleic acids, carbohydrates and lipids.
In one embodiment, the biological component comprises a protein selected from the group consisting of a blood protein, a bacterial protein, a viral protein, a fungal protein, and a self-aggregating or amyloid forming protein.
In a further embodiment, the blood protein is selected from the group consisting of blood clotting proteins (e.g., fibrinogen, fibrin peptides, fibrin, transglutaminase substrates, thrombin), serum proteins (e.g., albumin and globulin), platelet proteins, blood cell glycoproteins, and haemoglobin.
In another embodiment, the bacterial protein is selected from the group consisting of a bacterial fimbrial protein (e.g CgsA from E. coli and AgfA from Salmonella), a bacterial toxin protein (e.g., toxins from Bacillus anthracis, Corynebacterium diphtheriae, Clostridium botulium), a bacterial cell surface protein (e.g., peptidoglycan, lipoproteins), and a bacterial spore protein (e.g., from Gram positive bacteria and having a similar sequence or overall structure to the proteins forming ribbon appendages in Clostridum taeniosporum, chaplin proteins, rodlin proteins).
In yet another embodiment, the viral protein is selected from the group consisting of a viral envelope protein, a viral capsid protein, and a viral core protein. Suitably, the viral proteins are from a bacteriophage virus (e.g., the MS2 and PP7 proteins), norwalk virus (e.g., capsid protein), rotavirus (e.g., VP2, VP6 and VP7 proteins), coronavirus (e.g., SARS S, E and M proteins), bluetongue virus (e.g., VP2 protein), human papillomavirus (e.g., viral major structural protein, L1), hepatitis B (e.g., small envelope protein HBsAg), Hepatitis C virus (e.g., core, E1 and E2 proteins), influenza virus (e.g., neuraminidase and haemagglutinin and matrix proteins), poliovirus (e.g., capsid VP0, 1 and 3 proteins), HIV (e.g., Pr55gag, envelope proteins) and dengue B virus (e.g., envelope (e) and pre-membrane/membrane (prM/M).
In another embodiment, the fungal protein is selected from the group consisting of hydrophobin proteins (e.g., SC3 from Schizophyllum commune, RodA/B from Aspergillus fumigates, and equivalent proteins from yeast), fungal spore proteins, hyphal proteins, mycotoxins, and fungal prions (e.g., Sup35, Het S, URE 2, Rnq1, New 1).
In yet another embodiment, the self-aggregating protein is selected from the group consisting of prions (e.g., PrPSc and PrPc, Sup35, Het S, Ure 2, Rnq1, New 1), prion mimetic proteins, amyloid fibrils, cell surface adhesins from floc forming and filamentous bacteria in activated sludge, beta amyloid protein, tau protein, polyadenine binding protein, herpes simplex virus glycoprotein B, lung surfactant protein C, CsgA protein from E. coli, AgfA protein from Salmonella species, bacterial fimbrial proteins, apolipoproteins (e.g., apolipoprotein A1), hydrophobins from fungal species (e.g., SC3 from Schizophyllum commune, RodA/B from Aspergillus fumigates), chaplins (e.g., Chps A-H from Streptomyces spp), rodlins (e.g., Rd1A and Rd1B from streptomyces spp), gram positive spore coat proteins (e.g., P29a, P29b, GP85 and a SpoVM analogue), and barnacle cement-like proteins (e.g., the 19 kDa protein from Balanus albicostatus, and the 20 kDa protein from Megabalanus rosa, and the novel calcite-dependent cement-like protein from Balanus albicostatus).
In another embodiment, the nucleic acid is selected from a DNA molecule and an RNA molecule. In a further embodiment, the nucleic acid is selected from single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), double-stranded DNA (dsDNA) or double-stranded RNA (dsRNA). In one embodiment, the nucleic acid is derived from neurological tissue.
In another embodiment, the carbohydrate is selected from the group consisting of exopolysaccharide, lipopolysaccharide (EPS/LPS, sometimes known as endotoxin) (e.g., from Legionella species, E. coli, Staphylococcus species, Streptococcus species, Pseudomonas species, Acinetobactor species, Campylobactor species, and Bacillus species), peptidoglycan, cell wall components of plants, fungi and yeast (e.g., chitin, lignin, glucan), mucin preparations, glycolipids (especially brain derived glycolipids), glycoproteins (e.g., cell surface glycoproteins, Eap1p), spore extracts (e.g., from Bacillus spp, Clostridal spp and other spore-formers), polysaccharides from yeast capsules, and invertebrate secretions (e.g., from molluscan gels).
In another embodiment, the lipid is selected from the group consisting of glycolipids (e.g., brain-derived glycolipids), gangliosides (e.g., neuronal cell gangliosides such as GT1b, GT1a and gangliosides of more general cell origin such as GM1), and plant oils and lipids.
In a further embodiment, the biological component is part of a biological matrix. In one embodiment, the indicator is covalently linked to the biological matrix. The biological matrix may be a mimetic of the sample that is to be treated. In one embodiment, the biological matrix comprises one or more components selected from the group consisting of proteins, lipids, nucleic acids, and carbohydrates, or fragments or derivatives thereof. In another embodiment, the biological matrix may comprise a mixture of proteins. In a further embodiment, the biological matrix may comprise one or more components selected from the group consisting of blood, serum, albumin, mucus, egg, neurological tissue, food, culled animal material, and a commercially available test soil. In yet another embodiment of the invention, the biological matrix comprises one or more components selected from the group consisting of fibrinogen, thrombin, factor VIII, CaCl2, and, optionally, albumin and/or haemoglobin.
In one embodiment of the invention, the thermostable kinase is covalently linked to the biological component. In another embodiment, the thermostable kinase is genetically or chemically cross-linked to the biological component. In a further embodiment, the biological component is linked to the thermostable kinase in the form of a fusion protein.
The indicators of the invention may be used to validate treatment processes designed to remove/inactivate a contaminant selected from the group consisting of a protein, a lipid, a carbohydrate and a nucleic acid.
The biological process indicator of the invention may further comprise an agent to stabilise the kinase, such as metal ions, sugars, sugar alcohols or gel-forming agents.
The indicator of the invention (including any biological matrix) may also be “fixed” by treatment with 70% ethanol or isopropanol. To achieve this, the indicator/matrix is incubated in 70% isopropanol for 30 minutes at room temperature. This mimics one of the commonly encountered processes which may increase the resistance of contaminating materials on surgical instruments, and therefore provides the indicator with an effective way of monitoring the removal of such materials.
The biological process indicator of the invention may be immobilised in or immobilised on a solid support. In one embodiment, the biological process indicator is immobilised in the solid support, or is immobilised on the solid support by chemical cross-linking or adsorption. The indicator may be attached to the solid support via the thermostable kinase, or via the biological component.
In one embodiment, the solid support is an indicator strip, a dip-stick or a bead, and, optionally, further comprises means to attach the solid support to a surface (such as a projection, recess or aperture for attachment of the solid support to a surface by means of a screw, nut and bolt, or clamp). In a further embodiment, the solid support is a matrix and the indicator is dispersed within the matrix.
In one embodiment of the invention, the enzyme used to form the biological process indicator is not a lichenase, a xylanase, a xylosidase, a formiltransferase, a Taq polymerase, an alpha-amylase, or a beta-glucosidase.
In yet another embodiment of the invention, there is provided a test soil comprising an indicator as described above.
Preparation of the Biological Indicator
The biological indicator of the invention may be prepared by covalently linking a thermostable kinase to an appropriate biological component. Any suitable method of covalent attachment known in the art may be used. In one embodiment, the thermostable kinase is genetically or chemically cross-linked to the biological component, and in one embodiment, the indicator is prepared as a fusion protein.
Chemical cross-linking may be achieved using a range of homo- and hetero-bifunctional reagents commonly used for cross-linking of proteins for the generation of enzyme conjugates or other related purposes. For example, in an indicator comprising fibrin as the biological component, the fibrin and the thermostable kinase may be derivatised with the addition of SPDP (Perbio) to primary amine groups. The thermostable kinase can then be reduced to generate a reactive thiol group and this is then mixed with the fibrin to produce covalent fibrin-thermostable kinase linkages.
The kinases can also be chemically cross-linked to carbohydrates, lipids or other glycoconjugates using heterobifunctional agents following treatment of the target carbohydrate with meta-periodate. The cross-linking may be achieved using a variety of chemistries as outlined in Example 23.
Alternatively, the indicator may be prepared as a fusion protein. This is achieved by fusing a synthetic gene encoding an appropriate thermostable kinase (e.g., the gene encoding AK from Sulfolobus acidocaldarius or Thermatoga neopolitana) to a gene encoding an appropriate biological component. Detailed protocols for the preparation of fusion protein indicators are given in the Examples (see e.g., Examples 10 & 13).
Kinase Enzymes for Use in the Biological Indicator
The kinase enzymes used in the indicators of the invention are capable of generating a signal that is detectable over an extremely wide range. Generally, the kinase is detected using a substrate comprising ADP which is converted to ATP, itself used to generate light, eg. using luciferin/luciferase, detected using a luminometer. The wide range makes the indicator particularly suitable for validation as the kinase remains detectable even after many logs reduction in amount/activity. For sterility, most national institutes regard a 6 log reduction in the amount or activity of a contaminant as required before sterility can be validated. The kinases used in the indicators of the invention offer the potential of validating reduction in the amount or activity of contaminants well beyond 6 logs, to 8 logs and more.
Any suitable kinase enzyme may be used as the reporter kinase in the present invention. In one embodiment, the reporter kinase is an adenylate kinase, acetate kinase or pyruvate kinase, or a combination thereof.
The reporter kinases used in the invention may have a variety of recognized tertiary structures, e.g., the kinase may be a trimeric or monomeric kinase. These tertiary structures may be associated with an improved stability of the kinase to conditions such as e.g., temperature, pH, chemical denaturants, or proteases.
In one embodiment, the reporter kinase is a microbial kinase derived from an organism selected from the group consisting of Pyrococcus furiousus, P. abyssi, P. horikoshii, P. woesii, Sulfolobus solfataricus, Sacidocaldarius, S. shibatae, Rhodothermus marinus, Thermococcus litoralis, Thermatoga maritima, Thermatoga neapolitana and Methanococcus spp. In another embodiment, the kinase is a Sulfolobus sp. kinase or a Thermotoga sp. kinase. In yet another embodiment, the kinase is a A. acidocaldarius kinase, A. fulgidus kinase, A. pernix kinase, A. pyrophilus kinase, B. caldotenax BT1 kinase, Bacillus species PS3 kinase, B. stearothermophilus 11057 kinase, B. stearothermophilus 12001 kinase, B. thermocatenulatus kinase, C. stercocorarium kinase, Methanococcus spp. Kinase, M. ruber kinase, P. abyssi kinase, P. furiosus kinase, P. horikoshii kinase, P. woesii kinase, R. marinus kinase, S. acidocaldarius kinase, S. shibatae kinase, S. solfataricus kinase, T. ethanolicus kinase, T. thermosulfurogenes kinase, T. celere kinase, T. litoralis kinase, T. aquaticus YT1 kinase, T. caldophilus GK24 kinase, T. thermophilus HB8 kinase, T. maritima kinase or a T. neapolitana kinase. In yet a further embodiment, the kinase is a T. litoralis kinase, T. maritima kinase, or a T. neapolitana kinase.
In one embodiment, the reporter kinase is thermostable. As well as being resistant to high temperatures, thermostable kinases are also found to be resistant to other biochemical and physical processes that routinely damage or destroy proteins or render them inactive, such as exposure to certain chemicals e.g., chaotropes, free-radical damage, detergents, extremes of pH, exposure to proteases, protein cross-linking, encapsulation within non-permeable or semi-permeable membranes or polymers, or irreversible immobilisation onto surfaces. (See for example: Daniel R M, Cowan D A, Morgan H W, Curran M P, “A correlation between protein thermostability and resistance to proteolysis”, Biochem J. 1982 207:641-4; Rees D C, Robertson A D, “Some thermodynamic implications for the thermostability of proteins”, Protein Sci. 2001 10:1187-94; Burdette D S, Tchernajencko V V, Zeikus J G. “Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity”, Enzyme Microb Technol. 2000 27:11-18; Scandurra R, Consalvi V, Chiaraluce R, Politi L, Engel P C., “Protein thermostability in extremophiles”, Biochimie. 1998 November; 80(11):933-41; and Liao H H., “Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents”, Enzyme Microb Technol. 1993 April; 15(4):286-92, all of which are hereby incorporated by reference in their entirety).
Examples of kinases suitable for use in the invention are set out in SEQ ID NO.s 1-32 below. In one embodiment, the kinases used in the invention have at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID Nos: 1-32.
Other examples of suitable reporter kinases may be found in WO00/46357 and WO2005/093085, which are hereby incorporated by reference in their entirety.
In one embodiment of the invention, kinase activity is detected using an ATP bioluminescent detection system. A standard luciferin-luciferase assay method can detect as little as 10−15 moles of ATP. By coupling an enzymatic amplification to the bioluminescent detection methods it is possible to detect as few as 10−20 moles of kinase.
Stabilisation of the Biological Indicator
A number of additives and changes to formulation that increase the stability of an enzyme, e.g., a kinase, to heat inactivation will be known to those familiar with the art.
The addition of stabilising agents such as sorbitol up to a concentration of 4M, or other polyols such as ethylene glycol, glycerol, or mannitol at a concentration of up to 2M may improve the thermostability of the enzyme. Other additives such as xylan, trehalose, gelatin may also provide additional stabilisation effects either individually or in combination. Addition of a range of divalent metal ions, most notably Ca2+, Mg2+ or Mn2+ may also improve stability of the enzyme.
Chemical modification of the enzymes can also be used to improve their thermal stability. Reductive alkylation of surface exposed amino groups by glyoxylic acid (e.g Melik-Nubarov (1987) Biotech lefts 9:725-730), addition of carbohydrates to the protein surface (e.g., Klibanov (1979) Anal. Biochem. 93:1-25) and amidation (e.g., Klibanov (1983) Adv. Appl. Microbiol. 29:1-28) may all increase the stability of the enzyme. Further methods including the use of chemical cross-linking agents and the use of various polymeric supports for enzyme immobilisation are also relevant methods for increasing the thermostability of enzymes (reviewed in Gupta (1991) Biotech. Appl. Biochem. 14:1-11).
Similar modifications are also relevant to the stabilisation of the indicator against other sterilisation processes such as hydrogen peroxide or ozone. In particular, processes where the access of the gaseous phase sterilant to the enzyme is restricted, for example by encapsulation in a suitable polymer or formulation with an additive to reduce penetration of the gas, will provide useful methods for increasing the stability of the enzyme if required.
Many of the treatments that are effective at increasing the thermal stability of enzymes are also relevant to the stabilisation against protease treatments, e.g., for the development of an indicator for the effective inactivation of TSE agents by protease treatment. In general, a protein that shows high levels of thermostability is likely to also show a high degree of stability for degradative processes such as denaturation or protease treatment (See for example: Daniel R M, Cowan D A, Morgan H W, Curran M P, “A correlation between protein thermostability and resistance to proteolysis”, Biochem J. 1982 207:641-4; Rees D C, Robertson A D, “Some thermodynamic implications for the thermostability of proteins”, Protein Sci. 2001 10:1187-94; Burdette D S, Tchemajencko V V, Zeikus J G. “Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity”, Enzyme Microb Technol. 2000 27:11-18; Scandurra R, Consalvi V, Chiaraluce R, Politi L, Engel P C., “Protein thermostability in extremophiles”, Biochimie. 1998 November; 80(11):933-41; and Liao H H., “Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents”, Enzyme Microb Technol. 1993 April; 15(4):286-92). Thermostable kinases therefore generally show a higher degree of stability to the actions of the protease treatments designed to inactivate TSE agents than might equivalent mesophilic kinases. Depending on the type of process used, a kinase can also be selected to favour other characteristics of the process. Thus for a protease treatment at alkaline pH the protocol tends towards the use of a thermostable kinase from a moderately alkalophilic organism such as P. furiosus, whereas a protease treatment at acidic pH might use a kinase from an acidophile such as S. acidocaldarius or S. solfotaricus. 
If required to improve the stability of the kinase indicator to protease treatment a number of other options exist. A number of these are the same as those described above for the stabilisation of the enzyme against heat treatment. For example, formulations containing sorbitol, mannitol or other complex polymers reduce the levels of inactivation of the enzyme on the indicator surface. In addition, treatments that specifically reduce the rate at which a protease substrate is degraded are particularly relevant to this application. For example, the formulation of the kinase in a solution containing up to around 10 mg/ml (a 10-fold excess compared to the preferred concentration of the indicator) of a suitable carrier protein such as casein or albumin, that acts as alternative substrate for the protease, will specifically reduce the rate of digestion of the kinase indicator. Similarly, the addition of free amino acids such as glycine, tyrosine, tryptophan or dipeptides to the formulation would provide a means of substrate level inhibition of the enzyme and reduce local inactivation of the kinase indicator.
Thermostable kinases produced by recombinant expression in bacteria can also be used in the present invention. The genetic modification of enzymes has been shown to provide significant increases in thermal stability and by analogy such mutations are also likely to significantly enhance the stability of the indicator enzymes in other processes such as protease treatment or gaseous phase “sterilisation”. The comparison of the thermostability of the kinase enzymes taken with the defined 3-D structure of the trimeric (archaeal) AKs (Vonrhein et al. (1998) J. Mol. Biol. 282:167-179 and Criswell et al. (2003) J. Mol. Biol. 330:1087-1099) has identified amino acids that influence the stability of the enzyme.
Genetically engineered variants of kinases showing improved thermostability are also used in the invention, and can be generated in a number of ways. Essentially these involve the specific site-directed mutagenesis of amino acids believed to form part of the central core packing region of the trimeric molecule and random “directed evolution” methods where the whole molecule is subjected to subsequent rounds of mutagenesis and selection/screening of molecules with improved properties. Specific modified enzymes are set out in SEQ ID NOs: 17-19 (several variants are embraced by each reference). These modifications outlined are based on a hybrid approach using a consensus based approach to define regions likely to influence the thermostability of the enzymes based on observed differences between structurally related molecules. This is followed by either defined changes to incorporate the amino acids that correlate with the best thermostability or a random replacement to incorporate every available amino acid at the positions defined as being essential for thermostability.
The stability/resistance of the indicators that bind to biological components that are part of a matrix may be improved by increasing the concentration of the biological component in the matrix, or by increasing the degree of cross-linking. By way of example, one of the indicators of the invention employs a fibrin-reactive peptide-kinase indicator to effect cross-linking into a biological matrix containing fibrin, e.g., a fibrin film. By altering the fibrin film, e.g., by increasing the concentration of fibrin, or by increasing its degree of cross linking, it is possible to significantly increase the resistance of the indicator to specific processes.
The resistance of indicators containing biological components such as Sup35 can be increased by promoting the fibrilisation of the indicators. This provides a molecule with greater physical stability, and may be relevant to monitoring the inactivation of agents such as prion proteins, which are believed to be multimeric in nature.
In one embodiment, the indicator is formulated in a carrier selected from the group consisting of sucrose (e.g., at up to 1% w/v), mucin (e.g., at up to 0.5% w/v), and albumin (e.g., at up to 1 mg/ml).
Solid Supports
The biological indicator of the invention may be attached to a variety of solid supports. The supports may be with or without chemical modifications and may comprise one or more indicators in a variety of formulations, depending e.g., on the requirements of the process to be validated. In one form the support is a plastic, wood, ceramic, glass, textile, steel or other metallic or polymer surface onto which the indicator is dried/cross-linked as a means of immobilisation. The support can be a polycarbonate, polystyrene or polypropylene strip or dipstick, optionally with a flattened surface, onto which the indicator is applied. An additional type of support with a porous surface for attachment of indicator is also particularly useful as an indicator for gaseous processes. Plastic, wooden, metallic or ceramic beads may also provide a valuable format for the solid support, again with specific relevance to monitoring gaseous processes. Such supports have advantages for certain applications, as they provide a significantly increased surface area for the attachment of the indicator. In a further embodiment, the solid support is a matrix and the indicator is dispersed within the matrix. In yet another embodiment, the matrix is a complex biological matrix.
Immobilisation of the Biological Indicator onto the Solid Support
The indicators of the invention may be bound onto the solid support using any of a wide variety of methods known in the art.
In one embodiment of the invention, the indicator is bound onto the solid support via standard protein adsorption methods as outlined below.
Binding of the indicator onto the solid support may be achieved by methods routinely used to link protein to surfaces, e.g., incubation of protein in 0.1M sodium bicarbonate buffer at about pH 9.6 at room temperature for about 1 hour. Alternatively, the protein is covalently coupled to the surface using any of a wide range of coupling chemistries known to those familiar with the art. For example, an adenylate kinase fusion protein (e.g., to Sup35) derivatised with SPDP (Pierce chemicals; using manufacturer's instructions), reduced with DTT to provide free sulfhydryl groups for cross-linking, is covalently attached to a polystyrene support with a maleimide surface. Plastic surfaces with such sulfhydryl-binding surfaces are well described in the literature. An added benefit of this method of coupling is that, if required, the enzyme can be cleaved from the support, e.g., by reduction with DTT or MESNA, to allow the assay to be carried out separately to any indicator support. The indicators described in this application have the property that their activity is retained upon derivatisation and cross-linking to such supports.
Alternatively, an amine reactive surface on a polystyrene or polycarbonate support is used, with a bifunctional cross-linking agent such as monomeric glutaraldehyde, to provide direct non-cleavable cross-linking of the kinase indicator via free amine groups on the protein. UV treatment can also be used to directly link the indicator to a suitable support. Steel surfaces can be treated in a similar way to plastic surfaces to mediate covalent attachment of the indicator.
A wide variety of protein cross-linking reagents are available from companies such as Pierce chemical company (Perbio). Reagents reactive to sulfhydryl, amino, hydroxyl and carboxyl groups are designed for coupling proteins but they can equally be used for cross-linking proteins to either naturally reactive or coated solid supports such as plastics, other polymers, glass and metals. Reactive chemistries are also available for cross-linking the enzymes to carbohydrates. For example, the reagents BMPH ((N-[ß-maleimidopropionic acid]hydrazide.TFA), KMUH ((N-[k-maleimidoundecanoic acid]hydrazide), and MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride) can be used to cross link the indicator containing either a free sulfhydryl in the form of a cysteine residue or a chemically derivatised protein reduced to generate a sulfhydryl reactive group, to carbohydrates. This may be particularly important for a solid support which is either a complex carbohydrate (e.g., paper, cellulose-based membranes, gels or resins) or can be coated or treated with a carbohydrate solution to generate a suitably reactive surface.
For each type of support, the indicator may be formulated in a solution that enhances binding and/or stabilises the bound protein. Such formulations include solutions containing up to 10% (w/v) sucrose, sorbitol, mannitol, cellulose, or polyethylene glycol (PEG). In addition, the indicator can be formulated as part of a gel that is applied to the surface or lumen of a suitable support. Examples include alginate, agar or polyacrylamide matrices.
The indicator may also comprise an agent to stabilise the indicator, and suitable stabilising agents are selected from metal ions, sugars, sugar alcohols and gel-forming agents.
To facilitate use of the indicator, the indicator may further comprise means to attach the solid support to a surface, such as a projection, recess or aperture for attachment of the support to a surface by means of a screw, nut and bolt or clamp.
Kits Comprising the Biological Indicator
In a second aspect of the invention, there is provided a kit for use in validating a treatment process in which the amount or activity of a contaminant in a sample is reduced, comprising:                (i) a biological process indicator according to the first aspect of the invention, and        (ii) a substrate for the thermostable kinase.        
To carry out measurement of the kinase amount/activity, the kit can include means for detecting ATP, e.g., luciferin/luciferase and optionally a luminometer. In one embodiment, the substrate for the thermostable kinase is ADP.
From previous testing with known contaminants, data correlating the reduction in the amount or activity of the contaminant with kinase activity can be prepared, and the kit therefore can also include one or more look-up tables correlating kinase activity with the reduction in amount or activity of a list of specified contaminants. In one embodiment, the kit is for monitoring TSE inactivation. In a further embodiment, the kit is used for monitoring norovirus inactivation.
Use of the Biological Indicator
In a third aspect, the invention provides for the use of a thermostable kinase covalently linked to a biological component as a biological process indicator for validating a treatment process for reducing the amount or activity of a contaminant in a sample.
In one embodiment, the biological process indicator is formulated according to the first aspect of the invention.
In a particular use of the invention, an indicator according to the first aspect of the invention is the reporter in a method of indicating the possible presence of a contaminant (e.g., an infectious agent) following a cleaning or inactivation procedure. First, a sample containing the indicator is exposed to a cleaning/inactivation procedure (e.g., one or more of a selected temperature, pH or protease concentration). The next step is to remove any contaminating enzymatic activity by heat treatment, e.g., at from 60° C. to 80° C. for at least 10 minutes (i.e. under conditions that do not significantly affect the thermostable kinase). The indicator is then reacted at a temperature of between 30° C. and 70° C. with a substrate (e.g., ADP) to allow the generation of ATP. The formation of ATP can be measured by bioluminescent detection using luciferin/luciferase and a suitable luminometer at 20° C.-30° C. for 10 minutes to 1 hour. The light output reading from the luminometer gives a reading of the residual kinase activity, i.e. the activity of the kinase following exposure to the cleaning/inactivation treatment. Based on data that have been previously derived from separate experiments, the method is completed by correlating the residual kinase activity with the possible presence of a contaminant within the treated sample.
In one embodiment, contaminating enzymatic activity or ATP in a sample may be removed by an initial treatment step (e.g., a selected temperature, pH or protease concentration), prior to addition of the indicator.
The use of the indicator of the invention to monitor/validate a variety of processes is now described.
In one embodiment, the indicator is used to validate the performance of a biological washing preparation in a wash cycle. Whilst validation of a wash cycle would potentially be of use in a domestic setting, its most advantageous use would be within a healthcare, pharmaceutical or food preparation setting, e.g., for validating decontamination of bedclothes, gowns or other items associated with patients suffering or exposed to infectious agents (e.g., an outbreak of methicillin resistant Staphylococcus aureus (MRSA) or Norwalk/Norwalk-like virus). In this context, the indicator of the invention has the advantage that it is relevant to biological material such as blood or other bodily fluids.
For the validation of a wash cycle, the indicator may be cross-linked onto a flexible wand, strip of cloth or other material suitable for inclusion within the cycle. The indicator is put into the washer with the remainder of the load. In one embodiment, the indicator may be fixed within a suitable holder on the inside of the washer to facilitate its recovery.
The wash cycle is then performed and the indicator removed and assessed prior to any further handling or processing of the load, using a “reader” which has been calibrated to indicate an acceptable level of residual kinase activity within the indicator—the acceptable level having been derived from previous calibration and assessment of suitable wash performance within the process. Such assessment might include the overall levels of soiling and the viable count of micro-organisms as assessed using suitable model organisms known to those familiar with the art. Based on the calibrated read-out, the load is passed for further processing or the wash cycle is repeated.
In a second embodiment, the indicator is used to validate processes for the inactivation of viruses. The detection of live viral isolates in the environment is problematic, particularly when associated with an emergency situation where speed and accuracy may be critical. The present invention provides the possibility of developing indicator systems that allow the monitoring of decontamination procedures essentially in real time. This would be particularly valuable for surface decontamination in healthcare and related facilities following either an outbreak (e.g., of Norwalk-like viruses) or a deliberate release of a viral agent (such as small pox).
An indicator for validating a viral inactivation process can take a variety of different forms, e.g., a wand or dipstick for monitoring an area sprayed or immersed with virucide, or a suspended indicator for monitoring a gaseous phase decontamination process. Alternatively, the indicator can be sprayed onto a surface prior to decontamination and the levels of residual kinase activity subsequently assessed by swabbing of the surface.
In a further embodiment of the invention, the indicator is used for validating protease degradation of bacterial protein toxins, plant toxins such as ricin, and other toxic proteins, peptides, or peptide analogues.
Proteases show significant potential for the degradation of a wide range of protein toxins that are potential biowarfare/bioterror threat agents including botulinum toxin, anthrax toxins and ricin. They also have the potential to inactivate a wide range of other potentially toxic or harmful protein or peptide agents to enable decontamination of surfaces/facilities or the safe disposal of materials. In this context, the indicator of the invention, together with the surface/material to be decontaminated, is subjected to the protease decontamination procedure. At the end of the procedure, the residual kinase activity of the indicator is assessed according to the method of the invention. The level of residual kinase activity is then correlated with inactivation indices for the particular protein toxin, or group of toxins. Assuming the level activity is equal to or below the defined index value then the material can be safely disposed of or the surface/facility returned to use.
In one embodiment, a suitable safety margin is built into the calibration of the inactivation indices to allow for any variability of the process performance. The additional stability of the enzymes used in this invention allow for this to be done with more certainty and greater dynamic range than a wide range of other enzymatic indicators, including those from “thermostable” organisms such as Bacillus stearothermophilus, as shown by the data showing the relative thermal stability of AKs from thermophilic organisms (FIGS. 1A, 1B, and 1C).
The indicator may also be used to validate protease decontamination procedures for cleaning down pharmaceutical production apparatus. A wide variety of pharmaceutical products use materials from either humans, or animals that might be contaminated with a wide variety of agents including prion (TSE) agents and viruses (e.g., West Nile virus, hepatitis, HIV). The risks may be exacerbated when the source of the material is of animal origin (e.g foetal calf serum, horse immunoglobulins) and where an intermediate processing stage may carry the risk of increasing the concentration of unidentified pathogens in a particular sample. The possibility of using a protease to clean down manufacturing facilities and apparatus (e.g., chromatography columns, vessels, pipework) between manufacturing batches has the potential to reduce or eliminate such risks, even when the contaminant has not been formally identified. This is particularly true for prion agents in, for example, blood fractionation apparatus where there is a significant risk of accumulation and of carrying an infection risk into the final product.
For validating this type of procedure, the indicator of the invention is ideally formulated as a dipstick to be immersed in the protease treatment solution, or as a cartridge to be attached in line with the apparatus to be cleaned. By assessing the levels of residual kinase activity in the indicator device following the treatment, and correlating this with the acceptable levels of cleaning, a rapid and reliable monitor of performance can be developed.
In another embodiment of the invention, the indicator is used for validating gas phase inactivation of contaminants, such as TSE.
The potential of ozone or other gas phase sterilants to inactivate such contaminants is suggested by a wide range of publications and articles, however, as yet, no method has explicitly been shown to be effective. To support the development and introduction of this gas phase technology into healthcare, a means of validating the performance of the technology will be required. As agents such as TSE have already been shown to be far more resistant to this form of inactivation than conventional viral or bacterial agents, the methods currently available for validating gas phase inactivation are unlikely to be suitable. The present invention addresses this problem.
For this type of validation, the indicator is attached onto a solid support by any suitable method, e.g., general adsorption and chemical cross-linking via amide, peptide, carbonyl, or cysteine bonds. For example, for ozone sterilisation, a rigid polyvinyl chloride (PVC), glass, steel, polyamide or polypropylene support may be used, with the indicator coupled to the support by any one of the methods previously described. The indicator is then included in the batch of material/instruments to be sterilised, exposed to the ozone, and assessed against a suitably calibrated inactivation index designed for assessing corresponding inactivation of the agent in question. Successful inactivation allows onward processing or use of the material/instruments.
The indicator may optionally be attached to the internal face of a tube or equivalent internal space, such that the penetration of the gas is restricted. This provides for a monitor that is suitable for assessing the penetration of the gas into equivalent spaces in instruments with lumens, or through packed loads of material. Alternatively, the indicator may be attached to porous materials such as polystyrene beads, or may be immobilised within a gel or resin.
In a further embodiment of the invention, the indicator is used for validating liquid chemical sterilisation systems (e.g., ENDOCLENS-NSX™) as used for processing of endoscopes and related equipment.
A wide range of endoscopes are routinely used in medicine and are an important part of medical diagnosis and treatment. These instruments are extremely sensitive and have posed a very significant problem for routine cleaning and disinfection. Traditionally, and remaining in current practice, endoscopes are cleaned by hand before being decontaminated using a low temperature method. A range of chemical disinfectants and automated re-processing apparatus has been developed to address the specific issues of decontaminating sensitive pieces of equipment such as endoscopes, where traditional autoclaving is not possible. These methods have helped to reduce the levels of contamination on difficult to clean instruments, which have been associated with the iatrogenic transmission of a wide range of viral and bacterial pathogens. The current method of validating such processes is to monitor the flow rate and temperature of the washing solution. The indicator of the invention provides for a further means of validation that provides a read-out of actual cleaning effectiveness within the endoscope lumen.
For this type of validation, the indicator is attached to the internal surface of a tube designed to be of a similar overall internal diameter to the endoscope tube. This indicator apparatus is connected in series to the endoscope on the automatic reprocessing apparatus. The endoscope is then processed in the normal way. At the end of the process, preferably before the endoscope is removed from the apparatus, the indicator is detached and assessed for the level of kinase activity remaining. The level of activity may be correlated with previously defined thresholds for the acceptable performance of the process and, based on this assessment, the endoscope may be transferred for additional cleaning or decontamination or prepared for use. If the level of performance is not adequate then the instrument may be re-processed (using the same or more stringent conditions) with a new indicator attached as previously. The indicator apparatus is also suitable for validating the manual cleaning of endoscope and/or any other instrument with a lumen.
In a further embodiment of the invention, the indicator is used to monitor routine cleaning performance in washer-disinfectors, such as those used in hospitals.
In another embodiment of the invention, the indicator is used for monitoring glutaraldehyde or ortho-phthaldehyde (OPA) treatments. Glutaraldehyde and formaldehyde have been widely used as sterilants over many years. The chemical disinfectants work by multiply crosslinking proteins in a non-specific fashion to destroy their function. Ortho-phthaldehyde (OPA) has emerged recently as a new disinfectant in this family and is being widely used as it avoids some of the toxicity problems associated with glutaraldehyde. The indicator of the invention is suitable for the monitoring of all of this class of chemical disinfectants as the kinases are sensitive to non-specific cross-linking of this kind. The indicator may be covalently attached to a suitable surface and exposed to the chemical sterilant along with the other items to be sterilised. The effectiveness of the process is assessed by measuring the residual enzyme activity of the indicator. This activity is compared to defined threshold values that indicate the correct performance of the process.
The use of different types of kinase may provide additional sensitivity or susceptibility to the process as may be required for different applications. The thermostable adenylate kinases described in this specification can be broadly classified into two groups based on their molecular architecture. Thus, the enzymes from Sulfolobus species are examples of enzymes that have a trimeric structure with a central hydrophobic core that is the principle determinant in maintaining their activity at high temperatures. The second group of enzymes are monomeric, exemplified by the adenylate kinases from Thermatoga species, but have a slightly longer polypeptide chain with an additional “lid” domain that affects the active site. These different types of thermostable enzymes will show differential sensitivity to this type of chemical sterilant due to the variable flexibility of their peptide chains during enzyme action. For any particular sterilant and/or concentration an empirical screen will identify enzymes with suitable susceptibilities for monitoring and validating these types of chemicals.
In a further embodiment of the invention, the indicator is used as an ultra-rapid read-out monitor for ethylene oxide, hydrogen peroxide or other gas phase processes.
A wide range of gas phase sterilants are currently being used by a variety of manufacturers for routine disinfection of bacterial and viral agents. The current methods exploit the oxidative properties of the gases to destroy peptide linkages. As such, the kinases of the present invention, with their robust physicochemical properties, are ideal for providing a very rapid read-out of inactivation. The indicator in this example is similar to those described previously, e.g., in relation to the ozone inactivation of agents such as TSE.
A particularly challenging issue for sterilisation and decontamination processes is the ability to validate sterility of large bulk liquids, as might be required in the manufacture of various medicines or other pharmaceutical products. Whilst current methods monitor the temperature, time, and/or pressure parameters of a particular process (depending on its precise nature), there are few, if any, available methods for validating actual sterilisation within the bulk liquid. This is difficult even within volumes of around 1 liter, but is almost impossible at larger volumes.
The present invention provides a number of possible solutions to address this problem. In its simplest form, the indicator may be added to the liquid to be sterilised at a concentration suitable for measuring defined levels of kinase inactivation at the end of the process and equating this to levels of sterilisation. Whilst this might not be desirable in certain types of processes, the inert nature of the kinase and the ubiquitous presence of equivalent enzyme activities in all organisms, may make it acceptable. The acceptability may be improved by the fact that many thermostable enzymes are highly condensed and thus have very low immunogenicity following inoculation into animals.
Where such direct additions are not acceptable, the indicator may be added to the bulk liquid in a dialysis sack, porous container or immobilised to a suitable support such that no part of the indicator is released into the bulk liquid, but the sterilising conditions work on the indicator in the same way as for the whole sample. A wide variety of possible ways of containing or immobilising proteins, to allow general diffusion of the liquid sample but to restrict the movement of the indicator sample, will be known to those familiar with the art. Possible examples include, but are not limited to dialysis membranes, Visking tubing, porous membranes, protein-binding resins, rigid gels or solid supports as described for the other indicators discussed. The indicator may be attached to the surface by any one of the methods discussed previously, or simply encased within a suitable membrane without attachment, such that the indicator may be simply removed from the bulk liquid at completion of the process.
Method of Validating a Treatment Process
In a fourth aspect, the invention provides a method of validating a treatment process for reducing the amount or activity of a contaminant in a sample, comprising the steps of:                (a) obtaining a sample that contains or is suspected to contain a contaminant;        (b) subjecting the sample to a treatment process in the presence of a defined amount of a thermostable kinase covalently linked to a biological component;        (c) measuring residual kinase activity and optionally calculating the reduction in kinase activity; and        (d) comparing said residual kinase activity to a predetermined kinase activity, or comparing said reduction in kinase activity to a pre-determined reduction in kinase activity, wherein the pre-determined kinase activity or the pre-determined reduction in kinase activity corresponds to a confirmed reduction in the amount or activity of the contaminant under the same conditions.        
It is possible that the sample in step (a) may not contain any contaminant at all. The point of the validation is that, after carrying out the treatment, it is confirmed that any agent that might have been present has been removed/inactivated to an acceptable degree. In general, however, the sample is known to contain, or suspected to contain, the contaminant.
In one embodiment, the thermostable kinase used in step (b) of the method is formulated as an indicator according to the first aspect of the invention.
In another embodiment, the residual kinase activity in step (c) is measured by adding a substrate comprising ADP to the residual kinase and measuring the formation of ATP. ATP formation can be measured by bioluminescent detection using luciferin/luciferase and a suitable luminometer.
Typically, an operator measures kinase activity before and after treating the sample. It is also possible that contaminating, usually mesophilic, kinase can get into the sample prior to assaying for kinase activity. Thus, in one embodiment of the invention, the assay includes the step of inactivating mesophilic kinase, such as by treating the sample at 70° C. for at least 30 minutes, or at 80° C. for at least 10 minutes, prior to measuring residual kinase activity.
In one embodiment, the kinase, prior to the treatment, has an activity of at least 10,000,000 Relative Light Units (RLU) per mg kinase, or at least 8,000,000 RLU per mg kinase, or at least 5,000,000 RLU per mg kinase, or at least 3,000,000 per mg kinase, or at least 1,000,000 RLU per mg kinase, or at least 500,000 RLU per mg kinase, when measured in the presence of luciferin/luciferase by a luminometer.
In another embodiment of the invention, the predetermined kinase activity is less than 10,000 RLU per mg kinase, or less than 1000 RLU per mg kinase, or less than 500 RLU per mg kinase, or less than 250 RLU per mg kinase, or less than 100 RLU per mg kinase, or less than 10 RLU per mg kinase, or less than 1 RLU per mg kinase, or is 0 RLU per mg kinase.
In a further embodiment of the invention, the predetermined reduction in kinase activity is equal to or greater than a 1 log reduction, or a 2 log reduction, or a 3 log reduction, or a 4 log reduction, or a 5 log reduction, or a 6-log reduction, or a 7 log reduction, or an 8 log reduction or a 9 log reduction in kinase activity.
In another embodiment, the predetermined reduction in kinase activity corresponds to a 3 log reduction, or a 6 log reduction, or a 7 log reduction, or an 8 log reduction, or a 9 log reduction, in the amount or concentration of the kinase. In further embodiments, the predetermined reduction in kinase activity corresponds to a reduction in RLU of at least 800,000, or at least 900,000, or at least 950,000, or at least 990,000, or at least 999,000, or at least 999,900, or at least 999,990, or at least 999,999 RLU.
In yet another embodiment of the invention, the confirmed reduction in the amount or activity of the contaminant within the sample is at least 3 logs, at least 6 logs, ably at least 7 logs, more ably at least 8 logs, most ably at least 9 logs.
In another embodiment of the invention, the treatment is continued until the residual kinase activity or the reduction in the kinase activity corresponds to a confirmed reduction in the amount or activity of the contaminant of at least 3 logs, at least 6 logs, or at least 7 logs, or at least 8 logs, or at least 9 logs.
In one embodiment of the invention, the method further comprises the step of recording the data obtained in step (c) on a suitable data carrier.
Method of Correlating
In a fifth aspect, the invention provides a method of correlating the reduction in the amount or activity of a contaminant in a sample with the kinase activity of a biological process indicator as described in connection with the first aspect of the invention. This method comprises:                (i) preparing a sample containing a defined amount of the contaminant and a sample containing a defined amount of the indicator according to the first aspect of the invention, or preparing a single sample containing both a defined amount of the contaminant and a defined amount of the indicator according to the first aspect of the invention;        (ii) subjecting the sample or samples to a treatment;        (iii) measuring the residual activity of the indicator kinase and optionally calculating the reduction in kinase activity;        (iv) measuring residual amount or activity of the contaminant and optionally calculating the reduction in the amount or activity of the contaminant;        (v) repeating steps (i) to (v), wherein at least one of the treatment parameters is changed.        
In one embodiment, the treatment parameter comprises one or more of time, temperature, pH, pressure, protease concentration, and concentration of sterilant or detergent.
In a particular embodiment, the treatment comprises heating the sample(s) at 50-140° C., or 80-100° C., or 134-138° C.; the treatment parameter is time; and steps (i) to (iv) are repeated by subjecting the sample(s) to said treatment for periods of 1, 5, 10, 20, 40 and 60 minutes.
In a further embodiment, the treatment comprises exposing the sample(s) to a pH of 9-14, or pH 12 or above, or about pH 12; the treatment parameter is time; and steps (i) to (iv) are repeated by subjecting the sample(s) to said treatment for periods of 1, 5, 10, 20, 40 and 60 minutes.
In another embodiment, the treatment comprises exposing the sample(s) to a protease at a concentration of 0.5-2 mg/ml, or about 1 mg/ml, or about 2 mg/ml; the treatment parameter is time; and steps (i) to (iv) are repeated by subjecting the sample(s) to said treatment for periods of 1, 5, 10, 20, 40 and 60 minutes.
The above method enables preparation of calibration data for future use of the indicator for validation of a treatment on samples containing, or suspected of containing contaminant. The calibration of a number of treatment processes is described in WO2005/093085.